Research

CHLOROPLAST DIVISION IN PLANTS


The plastids of plant cells comprise an essential, metabolically diverse group of eukaryotic organelles. The most well-studied plastids are the green chloroplasts, which proliferate in leaf cells and carry out the life-sustaining process of photosynthesis. Plastids also synthesize many other compounds critical for plant growth and development, including membrane lipids, amino acids and growth regulators, and consequently must be partitioned to daughter cells during cell division. Further, specialized plastid types manufacture products of major agricultural importance, such as oil and starch important for both food and biofuels production. Recently, chloroplasts have also been exploited as factories for the production of biopharmaceuticals. Plastid propagation in dividing cells and the developmentally regulated proliferation of specific plastid types in different plant organs depend on the process of plastid division. Thus, plastid division is a fundamentally critical aspect of plant development with important implications for agriculture and biotechnology.

Our research centers on elucidating the mechanisms powering plastid division in plant cells. This process is orchestrated by a dynamic macromolecular machine composed of several ring-shaped sub-complexes that function in concert to constrict the organelle (Fig. 1A).  The recruitment, assembly and biochemical activities of these subcomplexes must be coordinated across the two envelope membranes to achieve organelle fission (Fig. 1B).  We are combining the powerful genetic and genomic resources of Arabidopsis with the tools of biochemistry and cell biology to identify the components of the plastid division machinery, define their functions within the division complex, and discover how chloroplast division is regulated. Because key features of plastid division are derived from the cell division machinery in the cyanobacterial endosymbiont from which chloroplasts evolved, we have also strategically incorporated experiments on cyanobacterial cell division into our work, and are pursuing comparative genomic and other computational strategies to uncover the full network of genes and proteins controlling plastid division in plants.
 

 

 

The plastid division machinery
Fig. 1. The plastid division machinery. (A) The tubulin-like FtsZ ring (Z-ring), inner plastid-dividing ring, dynamin-like ARC5 ring and outer plastid-dividing ring assemble sequentially and function together to constrict and sever the envelope membranes.  The inner and outer plastid-dividing rings are distinct from the FtsZ and ARC5/dynamin rings (reviewed by Osteryoung and Nunnari, 2003; Yang et al., 2008; Miyagishima, 2010, Plant Physiol. 155: 1533).  Recently, the outer PD ring was shown to be composed of polyglucan fibrils (Yoshida et al., 2010, Science 329: 949), but the inner PD ring composition is unknown. (B) Working model of the coordinated division machinery in Arabidopsis showing the topologies, interactions and hypothesized functional relationships among a subset of plastid division proteins.  Formation of the stromal Z-ring, composed of FtsZ1 (Z1) and FtsZ2 (Z2), occurs first. Z-ring assembly and dynamics are antagonistically regulated by ARC6, which promotes assembly through direct interaction with FtsZ2, and PARC6, which promotes FtsZ disassembly through ARC3.  Once the Z-ring is established, ARC6 interacts directly with and recruits PDV2 to the division site, and PARC6 recruits PDV1 to the division site.  PDV1 and PDV2 recruit ARC5 independently from the cytosol to the division site, but both PDV1 and PDV2 are required for full ARC5 contractile activity.  The PD rings and other division components have been omitted for simplicity.  Many aspects of this working model remain to be elucidated.  IEM, inner envelope membrane; OEM, outer envelope membrane; IMS, intermembrane space; N, N-terminus; C, C-terminus.  Adapted from Glynn, Yang et al., 2009.

  
A sampling of  chloroplast division proteins studied in our laboratory (all nuclear-encoded):

FtsZ1 and FtsZ2: Tubulin-like proteins related to bacterial FtsZ, a cytoskeletal GTPase that forms a ring at the midcell during bacterial cytokinesis (reviewed by Erickson et al., 2010, Microbiol. Mol. Biol. Rev. 74:504).  In plants, FtsZ1 and FtsZ2 are endosymbiotic in origin and colocalize to a ring (the Z-ring) at the chloroplast division site inside the chloroplast stroma (Fig. 2).  FtsZ1 and FtsZ2 interact with different assembly regulators and both proteins are required for full plastid-division activity (Schmitz et al., 2009).  Mutations that reduce the levels of either FtsZ1 or FtsZ2 protein cause dose-dependent defects in chloroplast division (Fig. 3), suggesting their stoichiometry is important for their in vivo activities.  Consistent with this hypothesis, we have recently found that FtsZ1 and FtsZ2 preferentially coassemble in vitro as heteropolymers (Olson, Wang et al., 2010).  The plastidic FtsZ ring probably functions both to constrict the inner envelope membrane and to position other components of the division complex.
 

Immunofluorescence detection of FtsZ1 at the chloroplast division site in Arabidopsis
Fig. 2.  Immunofluorescence detection of FtsZ1 at the chloroplast division site in Arabidopsis.  Left, Nomariski DIC image of a constricted chloroplast.  Right, FtsZ localization in the same chloroplast.  3D reconstruction of an image stack shows the ring at the organelle periphery. FtsZ2 localizes identically (from Vitha et al., 2001).
Dose-dependent chloroplast division defects in Arabidopsis ftsZ1 antisense lines.
Fig. 3. Dose-dependent chloroplast division defects in Arabidopsis ftsZ1 antisense lines.  Single leaf cells are shown from wild type plants (left) and plants lacking some (middle) or nearly all (right) FtsZ1 protein (images from Osteryoung et al., 1998).




 

 
 ARC5*: A member of the dynamin family of large GTPases, which oligomerize to function as

Dubmbell-shaped chloroplasts in the arc5 mutant
Fig. 4. Dubmbell-shaped chloroplasts
in the arc5 mutant (from Gao et al., 2003).

membrane “pinchases.” ARC5 is localized at the division site on the cytosolic surface of the outer envelope membrane where it constricts chloroplasts from the outside.  Plants with mutations in ARC5 exhibit enlarged, dumbbell-shaped chloroplasts (Fig. 4). ARC5 has no obvious homologues in bacteria, indicating it is eukaryotic in origin. 
*ARC5 is also called DRP5B.



PDV1 and PDV2: Plant-specific proteins of the chloroplast outer envelope that recruit ARC5 from the

GFP-PDV1 localization in leaf cell chloroplasts
Fig. 5. GFP-PDV1 localization in leaf cell chloroplasts.
Red is chlorophyll auto-fluorescence (from Miyagishima et al., 2006).

cytosol to the surface of the chloroplast at the division site. PDV1 localizes to a punctate mid-plastid ring prior to and during constriction and persists as a spot at the pole of one of the daughter chloroplasts following division (Fig. 5). PDV2 also localizes to the chloroplast division site, but forms a continuous ring and is not retained at the pole following division (Glynn et al., 2008).

ARC6 and PARC6:  Proteins of the chloroplast inner envelope that both regulate FtsZ ring assembly and dynamics inside the chloroplast (Fig. 5) and position PDV1 and PDV2 to the mid-plastid division site in the outer envelope (Fig. 6), resulting in ARC5 recruitment to the chloroplast surface.  ARC6 is cyanobacterial in origin whereas its paralogue PARC6 is unique to vascular plants.  ARC6 and PARC6 have divergent functions, but both play critical roles in coordinating the contractile activities of the FtsZ and ARC5 rings across the two envelope membranes (Fig. 1B)

ARC6 localization and effect of ARC6 on chloroplast division and FtsZ filament morphology
Fig. 6.  ARC6 localization and effect of ARC6 on chloroplast division and FtsZ filament morphology.  (A) ARC6-GFP localization at the chloroplast division site in wild-type Arabidopsis. (B, C) Immunofluorescence labeling of FtsZ in chloroplasts of (B) an arc6 mutant and (C) a 35S-ARC6 overexpressor.  Chloroplasts in single leaf cells are shown and chloroplast shapes, severely enlarged in the arc6 mutant and ARC6 overexpressor, are visible in the faint background fluorescence (from Vitha et al., 2003).
ARC6 is required for PDV1 and PDV2 positioning
Fig. 7. ARC6 is required for PDV1 and PDV2 positioning. Merged images showing YFP or GFP (green) and chlorophyll autofluorescence (red) in leaf cells of wild type  and an arc T-DNA mutant. Arrows show midplastid YFP-PDV2 or GFP-PDV1 rings.  PDV1 and PDV2 are distributed diffusely over the chloroplast surface in the arc6 mutant (from Glynn et al., 2008).  Similar experiments have shown that PARC6 is required for PDV1 but not PDV2 positioning (Glynn, Yang et al., 2009). 

 

Selected Publications

 

Yoshida, Y., Y. Mogi, A.D. TerBush and K.W. Osteryoung. Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization. 2016. Nature Plants 2: 16095.

Zhang, M., C. Chen, J.E. Froehlich, A.D. TerBush and K.W. Osteryoung. 2016. Roles of Arabidopsis PARC6 in coordination of the chloroplast division complex and negative regulation of FtsZ assembly. Plant Physiol. 170: 250-262.

Larkin, R.M., G. Stefano, M.E. Ruckle, A.K. Stavoe, C.A. Sinkler, F. Brandizzi, C.M Malmstrom and K.W. Osteryoung. 2016. REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment. Proc. Natl. Acad. Sci. USA. 113: E116-E1125. 

TerBush, A.D., C.A. Porzondek and K.W. Osteryoung. 2016. Functional analysis of the chloroplast division complex using Schizosaccharomyces pombe as a heterologous expression system. Microsc. Microanal. 22: 275-289.   

Kumar, N., A. Radhakrishnan, C.-C. Su, K.W. Osteryoung and E.W. Yu. 2015. Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6. Protein Sci. 25: 523-529.

Dutta, S., J.A. Cruz, Y. Jiao, J. Chen, D.M. Kramer and K.W. Osteryoung. 2015. Non-invasive, whole-plant imaging of chloroplast movements and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants. Plant J. 84: 428-442.

Spitzer, C., F. Li, R. Buono, H. Roschzttardtz, T. Chung, M. Zhang, K.W. Osteryoung, R.D. Vierstra and M.S. Otegui. 2015. The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis. Plant Cell 27: 391-402.

Osteryoung, K.W. and K.A. Pyke. 2014. Plastid division and dynamic morphology. Annu. Rev. Plant Biol. 65: 443-472.

Zhang, M., A.J. Schmitz, D.K. Kadirjan-Kalbach, A.D. TerBush and K.W. Osteryoung. 2013. Chloroplast division protein ARC3 regulates chloroplast FtsZ-ring assembly and positioning in Arabidopsis through interaction with FtsZ2. Plant Cell 25: 1787–1802.

TerBush, A.D., Y. Yoshida and K.W. Osteryoung. 2013. FtsZ in chloroplast division: structure, function and evolution. Curr. Opin. Cell Biol. 25: 461-470.

TerBush, A.D. and K.W. Osteryoung. 2012. Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling. J. Cell. Biol. 199: 623-637.

Kadirjan-Kahlbach, D.K., D.W. Yoder. M.E. Ruckle, R.M. Larkin and K.W. Osteryoung. 2012. FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division. Plant J. 72: 856–867

Vieler, A., G. Wu, C. Tsai, B. Bullard, A. Cornish, C. Harvey, I. Reca, C. Thornburg, R. Achawanantakun, C.J. Buehl, M.S. Campbell, D. Cavalier, K.L. Childs, T.J. Clark, R. Desphande, E. Erickson, A.A. Fergusson, W. Handee, Q. Kong, X. Li, B. Liu, S. Lundback, C. Peng, R. Roston, S. Sanjaya, J. Simpson, A. TerBush, J. Warakanont, S. Zäuner, E. Farre, E. Hegg, N. Jiang, M. Kuo, Y. Lu, K.K. Niyogi, J. Ohlrogge, K.W. Osteryoung, Y. Shachar-Hill, B.B. Sears, Y. Sun, H. Takahashi, M. Yandell, S. Shiu, and C. Benning. 2012. Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet. 8: e1003064.

Yang, Y., T.L. Sage, Y. Liu, T. R. Ahmad, W.F. Marshall, S. Shiu, J.E. Froehlich, K.M. Imre and K.W. Osteryoung. 2011. CLUMPED CHLOROPLASTS 1 is required for plastid separation in Arabidopsis. Proc. Natl. Acad. Sci. USA. 108: 18530-18535.

Ajjawi, I., A. Coku, J.E. Froehlich, Y. Yang, K.W. Osteryoung, C. Benning, and R.L. Last. 2011. A J-like protein influences fatty acid composition of chloroplast lipids in Arabidopsis. PLoS One 6: e25368.

Vitha, S. and K.W. Osteryoung. 2011. Immunofluorescence microscopy for localization of Arabidopsis chloroplast proteins. Methods Mol. Biol. 774: 33-58.

Osteryoung, K.W. and A.P.M. Weber. 2011. Plastid biology: Focus on the defining organelle of plants. Plant Physiol. 155: 1475-1476.

Olson, B.J.S.C., Q. Wang, and K.W. Osteryoung. 2010. GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2. J. Biol. Chem. 285: 20634-20643.

Weber, A.P.M. and K.W. Osteryoung. 2010. From endosymbiosis to synthetic photosynthetic life. Plant Physiol., 154: 593-597.

Dong, G., Q. Yang, Q. Wang, Y. Kim, T.L. Wood, K.W. Osteryoung, A. van Oudenaarden and S.S. Golden. 2010. Elevated ATPase Activity of KaiC constitutes a circadian checkpoint of cell division in Synechococcus elongatus. Cell 40: 529-539.

Osteryoung, K.W. 2010. Classic paper nomination: A genetic analysis of chloroplast division and expansion in Arabidopsis—Commentary. Plant Physiol., doi: 10.1104/pp.110.160382.

Schmitz, A.J., J.M. Glynn, B.J.S.C. Olson, K.D. Stokes and K.W. Osteryoung. 2009. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development. Mol. Plant 2: 1211-1222.

Glynn, J. M., Y. Yang, S. Vitha, A.J. Schmitz, M. Hemmes, S. Miyagishima, and K.W. Osteryoung. 2009. PARC6, a novel chloroplast division factor, influences FtsZ assembly and is required for recruitment of PDV1 during chloroplast division in Arabidopsis. Plant J. 59: 700-711.

Suzuki, K., H. Nakanishi, J. Bower, D.W. Yoder, K.W. Osteryoung and S. Miyagishima. 2009. Plastid chaperonin proteins Cpn60α and Cpn60b are required for plastid division in Arabidopsis thaliana. BMC Plant Biology 9: 38-49.

Yang, Y., J.M. Glynn, B.J.S.C. Olson, A.J. Schmitz, and K.W. Osteryoung. 2008. Plastid division: across time and space. Curr. Opin. Plant Biol. 11: 577-84.

Glynn, J. M., J.E. Froehlich and K.W. Osteryoung. 2008. Arabidopsis ARC6 coordinates the division machineries of the inner and outer chloroplast membranes through interaction with PDV2 in the intermembrane space. Plant Cell 20: 2460–2470.

McAndrew, R.S., B.J.S.C. Olson, C.L. Chi-Ham, S. Vitha, J.E. Froehlich and K.W. Osteryoung. 2008. In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex. Biochem. J. 412: 367-378.

Lu, Y., L.J. Savage, I. Ajjawi, K.M. Imre, D.W. Yoder, C. Benning, D. Dellapenna, J.B. Ohlrogge, K.W. Osteryoung, A.P. Weber, C.G. Wilkerson and R.L. Last. 2008. New connections across pathways and cellular processes: industrialized mutant screening reveals novel associations between diverse phenotypes in Arabidopsis. Plant Physiol. 46:1482-500.

Shimada, H., M. Mochizuki, K. Ogura, J.E. Froehlich, K.W. Osteryoung, Y. Shirano, D. Shibata, S. Masuda, K. Mori, and K.I. Takamiya. 2007. Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase. Plant Cell 19: 3157-69.

Yoder, D.W., D. Kadirjan-Kalbach, B.J.S.C. Olson, S. Miyagishima, S.L. DeBlasio, R.P. Hangarter and K.W. Osteryoung. 2007. Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo. Plant Cell Physiol. 48: 775–791.

Glynn, J. M., S. Miyagishima and K.W. Osteryoung. 2007. Structure and dynamics of the chloroplast division complex. Traffic 8:451-561.

Miyagishima, S., J.E. Froehlich, and K.W. Osteryoung. 2006. PDV1 and PDV2 mediate recruitment of the dynamin-related protein arc5 to the plastid division site. Plant Cell 18: 2517-2530.

Gao, H., T.L. Sage and K.W. Osteryoung. 2006. FZL, an FZO-like protein in plants, is a determinant of thylakoid and chloroplast morphology. Proc. Natl. Acad. Sci. USA. 103: 6759-6764.

Miyagishima, S., C.P. Wolk and K.W. Osteryoung. 2005. Identification of cyanobacterial cell division genes by comparative and mutational analyses. Mol. Micro. 56: 126-143.

Osteryoung, K.W. and J. Nunnari. 2003. The division of endosymbiotic organelles. Science 302: 1698-1704.

Vitha, S., Froehlich, J.E., O. Koksharova, K.A. Pyke, H. van Erp and K.W. Osteryoung. 2003. ARC6 is a J-domain plastid division protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15: 1918-1933.

Gao, H., D. Kadirjan-Kalbach, J.E. Froehlich and K.W. Osteryoung. 2003. ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc. Natl. Acad. Sci. USA, 100: 4328–4333.

Froehlich, J.E., C.G. Wilkerson, W.K. Ray, R.S. McAndrew, K.W. Osteryoung, D.A. Gage and B.S. Phinney. 2003. Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. J. Proteome Res., 2: 42-425.

Stokes, K.D., and K.W. Osteryoung, 2003. Early evolutionary divergence of the FtsZ1 and FtsZ2 plastid division gene families in photosynthetic eukaryotes. Gene 320: 97-108.

Hong, Z., S.Y. Bednarek, E. Blumwald, I. Hwang, G. Jurgens, D. Menzel, K.W. Osteryoung, N.V. Raikhel, K. Shinozaki, N. Tsutsumi, D.P.S. Verma. 2003. A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions. Plant Mol. Biol. 53: 261-265.

Osteryoung, K.W. 2002. Chloroplast division: a work of ARTEMIS. Curr. Biol. 13: R844-R845.

Osteryoung, K.W. and R.S. McAndrew. 2001. The plastid division machine. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 315-333.

Vitha, S., R.S. McAndrew and K.W. Osteryoung. 2001. FtsZ ring formation at the chloroplast division site in plants. J. Cell Biol. 153: 111-119.

McAndrew, R.S., J.E. Froehlich, S. Vitha, K.D. Stokes and K.W. Osteryoung. 2001. Colocalization of plastid division proteins to the chloroplast stromal compartment establishes a new functional relationship between FtsZ1 and FtsZ2 in higher plants. Plant Physiol. 127: 1656-1666.

Osteryoung, K.W. 2001. Fission of eukaryotic organelles. Curr. Opin. Microbiol. 4: 639-646.

Moehs, C.P., L. Tian, K.W. Osteryoung and D. DellaPenna. 2001. Analysis of carotenoid biosynthetic gene expression during marigold petal development. Plant Mol. Biol. 45: 281-293.

Osteryoung K.W. 2000. Organelle fission: Crossing the evolutionary divide. Plant Physiol. 123:1213-1216.

Stokes K.D., R.S. McAndrew, R. Figueroa, S. Vitha and K.W. Osteryoung. 2000. Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant Physiol. 124: 1668-1677.

Colletti, K.S., E.A. Tattersall, K.A. Pyke, J.E.Froelich, K.D. Stokes, and K.W Osteryoung. 2000. A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus. Curr. Biol. 10: 507-16.

Osteryoung, K.W., K.D. Stokes, S.M. Rutherford, A.L. Percival and W.Y. Lee. 1998. Chloroplast division in higher plants requires members of two functionally divergent FtsZ gene families. Plant Cell 10: 1991-2004.

Osteryoung, K.W. and K. A. Pyke. 1998. Plastid division: evidence for a prokaryotically derived mechanism. Curr. Opin. Plant Biol. 1: 475-479.

Osteryoung, K.W. and E. Vierling. 1995. Conserved cell and organelle division. Nature 376: 473-474.